Dexmedetomidine Inhibits Hippocampal Neuronal Damage Caused by Persistent Isoflurane-Induced Hypotension in Rat Model of Chronic Cerebral Hypoperfusion.

Purpose The purpose of this study was to investigate the effect of dexmedetomidine (DEX) on hypotension-induced neuronal damage in a chronic cerebral hypoperfusion (CCH) model of rats, an established model of cerebral white matter lesions (WML) in humans, which is prevalent in the elderly and closely related to cognitive decline. Methods The CCH model rats were randomly assigned to one of four groups: normotension + no DEX (NN) group (n = 6), normotension + DEX (ND) group (n = 6), hypotension + no DEX (HN) group (n = 6), or hypotension + DEX (HD) group (n = 6). Under isoflurane anesthesia, mean arterial blood pressure was maintained at or above 80 mmHg (normotension) or below 60 mmHg (hypotension) for a duration of two hours. The DEX groups received 50 µg of DEX intraperitoneally. Two weeks later, the Y-maze test and, after preparing brain slices, immunohistochemical staining were performed using antibodies against neuronal nuclei (NeuN), microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and Ionized calcium-binding adapter molecule 1 (Iba1). Results Behavioral observations showed no significant differences among the groups. Significant reductions of both NeuN-positive cells and the MAP2-positive area were found in the hippocampal CA1 in the HN group compared with NN and ND groups, but not in the HD group. GFAP and Iba-1-positive areas were significantly increased in the HN group, but not in the HD group. Conclusion DEX significantly ameliorated hypotension-induced neuronal damage and both astroglial and microglial activation in the CA1 region of CCH rats.

UPF1 plays critical roles in early B cell development.

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.

Synthesis of Branch-Type 3-Allylindoles from N-Alkyl-N-cinnamyl-2-ethynylaniline Derivatives Using π-Allylpalladium Chloride Complex as a Catalyst.

The reaction of N-alkyl-N-cinnamyl-2-ethynylaniline derivatives 1 via annulation and aza-Claisen-type rearrangement easily afforded corresponding branch-type 3-allylindoles 2 with high regioselectivities in good yields using π-allylpalladium chloride complex as a catalyst.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

Formal [3 + 2] Cycloaddition of α-Imino Esters with Azo Compounds: Facile Construction of Pentasubstituted 1,2,4-Triazoline Skeletons.

1,2,4-Triazole and 1,2,4-triazoline are important components of bioactive molecules and catalysts employed in organic synthesis. Therefore, the efficient synthesis of these components has received significant research attention. However, studies on their structural diversity remain lacking. Previously, we developed chiral phase-transfer-catalyzed asymmetric reactions of α-imino carbonyl compounds with α,ß-unsaturated carbonyl compounds and haloalkanes. In this study, we demonstrate the formal [3 + 2] cycloaddition reaction of α-imino esters with azo compounds under Brønsted base catalysis, resulting in the corresponding 1,2,4-triazolines in high yields. The results revealed that a wide range of substrates and reactants can be applied, irrespective of their steric and electronic characteristics. The present reaction made the general preparation of 3-aryl pentasubstituted 1,2,4-triazolines possible for the first time. Furthermore, a mechanistic study suggested that the reaction proceeds without isomerization into the aldimine form.

Intramolecular Azo Coupling Reaction of Binaphthyl Compounds: Synthesis of Pyrazole-Containing Helicene-Like Molecules.

A method for accessing pyrazole-containing helicene-like molecules from easily accessible NOBIN derivatives was developed. The reaction proceeded efficiently via diazonium salt intermediates, which provided a series of helicene-like molecular products in yields of 77%-89% regardless of their steric and electronic natures. The photophysical properties of the products were investigated. The 3,3'-disubstituted molecules showed a characteristic blue shift in their emission spectra. Product derivatizations were conducted, and interesting reactivities toward nucleophiles were observed.

Axially chiral N-alkyl-N-cinnamoyl amide type P,olefin ligands for Pd-catalyzed reactions.

We synthesized N-alkyl-N-cinnamoyl amide type phosphine-olefin compounds 1 and found axial chirality in a C(aryl)-N(amide) bond in compounds 1 by HPLC analysis using a chiral stationary phase column. We successfully obtained enantiomeric isomers of 1 and demonstrated the use of (-)-1 for chiral ligands in Pd-catalyzed asymmetric allylic substitution reactions of allylic esters with indoles (up to 97% ee).

Chiral Bromonium Salt (Hypervalent Bromine(III)) with N-Nitrosamine as a Halogen-Bonding Bifunctional Catalyst.

There has been a great focus on halogen-bonding as a unique interaction between electron-deficient halogen atoms with Lewis basic moieties. Although the application of halogen-bonded atoms in organic chemistry has been eagerly researched in these decades, the development of chiral molecules with halogen-bonding functionalities and their utilization in asymmetric catalysis are still in the\ir infancy. We have previously developed chiral halonium salts with amide functionalities, which behaved as excellent catalysts albeit in only two reactions due to the lack of substrate activation abilities. In this manuscript, we have developed chiral halonium salts with an N-nitrosamine moiety and applied them to the Mannich reaction of isatin-derived ketimines with malonic esters. The study focused on our novel bromonium salt catalyst which provided the corresponding products in high yields with up to 80% ee. DFT calculations of the chiral catalyst structure suggested that the high asymmetric induction abilities of this catalyst are due to the Lewis basic role of the N-nitrosamine part. To the best of our knowledge, this is the first catalytic application of N-nitrosamines.

Modification of proteins with azobenzene crosslinkers using reversible covalent bonds.

Thiol-reactive reagents designed for the chemical modification of proteins cannot, in general, be used directly for the modification of intracellular targets because the presence of millimolar concentrations of glutathione inside cells effectively outcompetes reaction with target thiols. Here we report an equilibrium, entropic strategy for achieving target selectivity using a cyanoacrylate-based thiol-reactive cross-linker (BCNA) with two reactive sites. This compound exhibits ≳200-fold selectivity for reaction with target peptides and proteins containing appropriately spaced pairs of thiols, versus reaction with mono-thiols. Photo-isomerization of the azobenzene moiety of the cross-linker can be used to affect the conformation of the target peptide or protein. This approach suggests a general strategy for the chemical modification of intracellular peptide and protein targets.

DSP-crosslinking and Immunoprecipitation to Isolate Weak Protein Complex.

Detecting protein-protein interactions (PPIs) is one of the most used approaches to reveal the molecular regulation of protein of interests (POIs). Immunoprecipitation of POIs followed by mass spectrometry or western blot analysis enables us to detect co-precipitated POI-binding proteins. However, some binding proteins are lost during cell lysis or immunoprecipitation if the protein binding affinity is weak. Crosslinking POI and its binding proteins stabilizes the PPI and increases the chance of detecting the interacting proteins. Here, we introduce the method of DSP (dithiobis(succinimidyl propionate))-mediated crosslinking, followed by tandem immunoprecipitation (FLAG and HA tags). The eluted proteins interacting with POI can be analyzed by mass spectrometry or western blotting. This method has the potential to be applied to various cytoplasmic proteins. Graphical abstract.

Regnase-1 Prevents Pulmonary Arterial Hypertension Through mRNA Degradation of Interleukin-6 and Platelet-Derived Growth Factor in Alveolar Macrophages.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a type of pulmonary hypertension (PH) characterized by obliterative pulmonary vascular remodeling, resulting in right-sided heart failure. Although the pathogenesis of PAH is not fully understood, inflammatory responses and cytokines have been shown to be associated with PAH, in particular, with connective tissue disease-PAH. In this sense, Regnase-1, an RNase that regulates mRNAs encoding genes related to immune reactions, was investigated in relation to the pathogenesis of PH. METHODS: We first examined the expression levels of ZC3H12A (encoding Regnase-1) in peripheral blood mononuclear cells from patients with PH classified under various types of PH, searching for an association between the ZC3H12A expression and clinical features. We then generated mice lacking Regnase-1 in myeloid cells, including alveolar macrophages, and examined right ventricular systolic pressures and histological changes in the lung. We further performed a comprehensive analysis of the transcriptome of alveolar macrophages and pulmonary arteries to identify genes regulated by Regnase-1 in alveolar macrophages. RESULTS: ZC3H12A expression in peripheral blood mononuclear cells was inversely correlated with the prognosis and severity of disease in patients with PH, in particular, in connective tissue disease-PAH. The critical role of Regnase-1 in controlling PAH was also reinforced by the analysis of mice lacking Regnase-1 in alveolar macrophages. These mice spontaneously developed severe PAH, characterized by the elevated right ventricular systolic pressures and irreversible pulmonary vascular remodeling, which recapitulated the pathology of patients with PAH. Transcriptomic analysis of alveolar macrophages and pulmonary arteries of these PAH mice revealed that Il6, Il1b, and Pdgfa/b are potential targets of Regnase-1 in alveolar macrophages in the regulation of PAH. The inhibition of IL-6 (interleukin-6) by an anti-IL-6 receptor antibody or platelet-derived growth factor by imatinib but not IL-1ß (interleukin-1ß) by anakinra, ameliorated the pathogenesis of PAH. CONCLUSIONS: Regnase-1 maintains lung innate immune homeostasis through the control of IL-6 and platelet-derived growth factor in alveolar macrophages, thereby suppressing the development of PAH in mice. Furthermore, the decreased expression of Regnase-1 in various types of PH implies its involvement in PH pathogenesis and may serve as a disease biomarker, and a therapeutic target for PH as well.

Synthesis of 3-Allylindoles via Annulation of N-Allyl-2-ethynylaniline Derivatives Using a P,Olefin Type Ligand/Pd(0) Catalyst.

Annulation of N-allyl-2-ethynylaniline derivatives easily afforded the corresponding 2-substituted 3-allylindole derivatives in good to excellent yields using P,olefin ligand/palladium catalst systems.

Enhancement of Regnase-1 expression with stem loop-targeting antisense oligonucleotides alleviates inflammatory diseases.

Regnase-1 is an ribonuclease that plays essential roles in restricting inflammation through degrading messenger RNAs (mRNAs) involved in immune reactions via the recognition of stem-loop (SL) structures in the 3' untranslated regions (3'UTRs). Dysregulated expression of Regnase-1 is associated with the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here, we developed a therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was mediated by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding of Regnase-1 toward the SL structures in its 3'UTR. Regnase-1-targeting MOs not only enhanced Regnase-1 expression by stabilizing mRNAs but also effectively reduced the expression of multiple proinflammatory transcripts that were controlled by Regnase-1 in macrophages. Intratracheal administration of Regnase-1-targeting MOs ameliorated acute respiratory distress syndrome and chronic fibrosis through suppression of inflammatory cascades. In addition, intracranial treatment with Regnase-1-targeting MOs attenuated the development of experimental autoimmune encephalomyelitis by promoting the expansion of homeostatic microglia and regulatory T cell populations. Regnase-1 expression was inversely correlated with disease severity in patients with multiple sclerosis, and MOs targeting human Regnase-1 SL structures were effective in mitigating cytokine production in human immune cells. Collectively, MO-mediated disruption of the Regnase-1 self-regulation pathway is a potential therapeutic strategy to enhance Regnase-1 abundance, which, in turn, provides therapeutic benefits for treating inflammatory diseases by suppressing inflammation.

Cyclin J-CDK complexes limit innate immune responses by reducing proinflammatory changes in macrophage metabolism.

Toll-like receptor (TLR) stimulation induces glycolysis and the production of mitochondrial reactive oxygen species (ROS), both of which are critical for inflammatory responses in macrophages. Here, we demonstrated that cyclin J, a TLR-inducible member of the cyclin family, reduced cytokine production in macrophages by coordinately controlling glycolysis and mitochondrial functions. Cyclin J interacted with cyclin-dependent kinases (CDKs), which increased the phosphorylation of a subset of CDK substrates, including the transcription factor FoxK1 and the GTPase Drp1. Cyclin J-dependent phosphorylation of FoxK1 decreased the transcription of glycolytic genes and Hif-1α activation, whereas hyperactivation of Drp1 by cyclin J-dependent phosphorylation promoted mitochondrial fragmentation and impaired the production of mitochondrial ROS. In mice, cyclin J in macrophages limited the growth of tumor xenografts and protected against LPS-induced shock but increased the susceptibility to bacterial infection. Collectively, our findings indicate that cyclin J-CDK signaling promotes antitumor immunity and the resolution of inflammation by opposing the metabolic changes that drive inflammatory responses in macrophages.

Chiral Symmetry Breaking of Monoacylated Anhydroerythritols and meso-1,2-Diols through Crystallization-Induced Deracemization.

We developed a chiral symmetry breaking method for monoacylated meso diols. The X-ray crystal structure analysis of monoacylated 1,4-anhydroerythritols, meso cyclic diols with a cis configuration, revealed that the O-(p-anisoyl) derivative crystallized as a racemic conglomerate of the P21 21 21 crystal system. It was confirmed that the substrate racemized by intramolecular transfer of the acyl group in the presence of a catalytic amount of base. Evaporating the solvent gradually from the solution or Viedma ripening to promote crystallization-induced deracemization efficiently led to enantiomer crystals. These results provide the first successful example of asymmetric expression and amplification by deracemization of sugar derivatives without an external chemical chiral source. Furthermore, we applied this methodology to acyclic meso-1,2-diols. Three O-monoacylated substrates were successfully deracemized to 99 % ee by Viedma ripening. We also developed asymmetric desymmetrization of meso-1,2-diols by combining acylation and crystallization-induced deracemization.

Cinnamoyl amide type chiral P,olefin ligands for Pd-catalyzed reactions.

We synthesized cinnamoyl amide type chiral P,olefin ligand (S)-4. We successfully obtained separable diastereomers of 4d and demonstrated Pd-catalyzed asymmetric allylic substitution reactions of indoles using (S,aS)-4d as a chiral ligand with high enantioselectivities (up to 98% ee).

IRAK1-dependent Regnase-1-14-3-3 complex formation controls Regnase-1-mediated mRNA decay.

Regnase-1 is an endoribonuclease crucial for controlling inflammation by degrading mRNAs encoding cytokines and inflammatory mediators in mammals. However, it is unclear how Regnase-1-mediated mRNA decay is controlled in interleukin (IL)-1ß- or Toll-like receptor (TLR) ligand-stimulated cells. Here, by analyzing the Regnase-1 interactome, we found that IL-1ß or TLR stimulus dynamically induced the formation of Regnase-1-ß-transducin repeat-containing protein (ßTRCP) complex. Importantly, we also uncovered a novel interaction between Regnase-1 and 14-3-3 in both mouse and human cells. In IL-1R/TLR-stimulated cells, the Regnase-1-14-3-3 interaction is mediated by IRAK1 through a previously uncharacterized C-terminal structural domain. Phosphorylation of Regnase-1 at S494 and S513 is critical for Regnase-1-14-3-3 interaction, while a different set of phosphorylation sites of Regnase-1 is known to be required for the recognition by ßTRCP and proteasome-mediated degradation. We found that Regnase-1-14-3-3 and Regnase-1-ßTRCP interactions are not sequential events. Rather, 14-3-3 protects Regnase-1 from ßTRCP-mediated degradation. On the other hand, 14-3-3 abolishes Regnase-1-mediated mRNA decay by inhibiting Regnase-1-mRNA association. In addition, nuclear-cytoplasmic shuttling of Regnase-1 is abrogated by 14-3-3 interaction. Taken together, the results suggest that a novel inflammation-induced interaction of 14-3-3 with Regnase-1 stabilizes inflammatory mRNAs by sequestering Regnase-1 in the cytoplasm to prevent mRNA recognition.

Asymmetric Synthesis of Indoline from Achiral Phthalimide Involving Crystallization-Induced Deracemization.

Asymmetric synthesis was performed by combining the photochemical reaction of an achiral substrate followed by crystallization-induced deracemization. The results indicated that a fused indoline produced by photochemical intramolecular δ-hydrogen abstraction and cyclization of N-(5-chloro-2-methylphenyl)phthalimide crystallized as a racemic conglomerate. Since this substrate has an aminal skeleton, racemization involving a ring-opening and ring-closing equilibrium process occurred under suitable conditions. Efficient racemization was observed in acetone containing a catalytic base, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Crystallization-induced dynamic deracemization by Viedma ripening from racemic indoline was performed with an excellent enantioselectivity of 99 % ee. Furthermore, one-pot asymmetric synthesis of the indoline was achieved by the photochemical reaction of achiral phthalimide followed by continuous attrition-enhanced deracemization converging to 99 % ee of enantiomeric crystals. This is the first example of asymmetric expression and amplification by photochemical hydrogen abstraction and crystallization-induced dynamic deracemization.

Regnase-1-related endoribonucleases in health and immunological diseases.

Dynamic changes in gene expression are key factors in the development and activation of immune cells. RNA metabolism is one of the critical steps for the control of gene expression. Together with transcriptional regulation, mRNA decay by specific ribonucleases (RNases) plays a vital role in shaping gene expression. In addition to the canonical exoribonuclease-mediated mRNA degradation through the recognition of cis-elements in mRNA 3' untranslated regions by RNA-binding proteins (RBPs), endoribonucleases are involved in the control of mRNAs in immune cells. In this review, we gleam insights on how Regnase-1, an endoribonuclease necessary for regulating immune cell activation and maintenance of immune homeostasis, degrades RNAs involved in immune cell activation. Additionally, we provide insights on recent studies which uncover the role of Regnase-1-related RNases, including Regnase-2, Regnase-3, and Regnase-4, as well as N4BP1 and KHNYN, in immune regulation and antiviral immunity. As the dysregulation of immune mRNA decay leads to pathologies such as autoimmune diseases or impaired activation of immune responses, RNases are deemed as essential components of regulatory feedback mechanisms that modulate inflammation. Given the critical role of RNases in autoimmunity, RNases can be perceived as emerging targets in the development of novel therapeutics.